More Like this

1. Isothermal Amplification and Lateral Flow Nucleic Acid Test for the Detection of Shiga Toxin-Producing Bacteria for Food Monitoring

   https://doi.org/10.3390/chemosensors10060210  

   Petrucci, Sabrina; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K. (June 2022, Chemosensors)

   Foodborne bacteria have persisted as a significant threat to public health and to the food and agriculture industry. Due to the widespread impact of these pathogens, there has been a push for the development of strategies that can rapidly detect foodborne bacteria on-site. Shiga toxin-producing E. coli strains (such as E. coli O157:H7, E. coli O121, and E. coli O26) from contaminated food have been a major concern. They carry genes stx1 and/or stx2 that produce two toxins, Shiga toxin 1 and Shiga toxin 2, which are virulent proteins. In this work, we demonstrate the development of a rapid test based on an isothermal recombinase polymerase amplification reaction for two Shiga toxin genes in a single reaction. Results of the amplification reaction are visualized simultaneously for both Shiga toxins on a single lateral flow paper strip. This strategy targets the DNA encoding Shiga toxin 1 and 2, allowing for broad detection of any Shiga toxin-producing bacterial species. From sample to answer, this method can achieve results in approximately 35 min with a detection limit of 10 CFU/mL. This strategy is sensitive and selective, detecting only Shiga toxin-producing bacteria. There was no interference observed from non-pathogenic or pathogenic non-Shiga toxin-producing bacteria. A detection limit of 10 CFU/mL for Shiga toxin-producing E. coli was also obtained in a food matrix. This strategy is advantageous as it allows for timely identification of Shiga toxin-related contamination for quick initial food contamination assessments. 

   more » « less
2. Biofunctionalized Magnetic Nanoparticles with Multiplex Touchdown PCR for Simultaneous and Rapid Detection/Identification of Campylobacter jejuni and Campylobacter coli

   https://doi.org/10.1155/2022/5104187  

   Wenbap, Pattarapong; Rattanarojpong, Triwit; Khunrae, Pongsak; Luangtongkum, Taradon; Erickson, Larry E.; Hansen, Ryan R.; Tuitemwong, Pravate (October 2022, Journal of Nanomaterials)

   Parvinzadeh Gashti, Mazeyar (Ed.)

   The simple, accurate, and rapid detection of foodborne pathogens is essential for public health. Development of an immunomagnetic separation (IMS) multiplex touchdown PCR (IMS–multiplex TD–PCR) assay for simultaneous detection and distinguishing of C. jejuni and C. coli is reported herein. Polyclonal antibody (pAb) against multiepitope antigen (MEA) was conjugated to ferromagnetic nanoparticles (FMNs) to produce anti-MEA FMNs. Optimal anti-MEA FMNs loading yielded 26.7 μg of immunoglobulin G (IgG) molecules per mg of FMNs with an average size of 72 ± 9  nm, corresponding to an 83% rate of pAb conjugation. Anti-MEA FMNs (20 μg) for IMS captured culturable C. jejuni cells at 3.54 × 10 2 colony-forming unit (CFU)/mL in pure culture, while higher amounts (40 and 60 μg) reduced the recovery. The scanning electron microscope (SEM) analysis revealed the attachment of anti-MEA FMNs to target bacteria, forming aggregated cells and magnetic nanoparticles in ellipse-like shapes. The subsequent multiplex TD–PCR assay simultaneously detected and distinguished C. jejuni and C. coli at 104 CFU/mL in mixed culture and at 103 CFU/mL for each individual species. Furthermore, the limit of detection (LOD) of the IMS–multiplex TD–PCR assay was 104 CFU/g in spiked chicken breast samples. Specificity was 100% for both C. jejuni and C. coli as none of the amplicons were detected in control samples where Campylobacter was absent. This assay is able to detect and distinguish C. jejuni and C. coli simultaneously and is simple, accurate, and rapid with a time to result of 4 h without an enrichment step, making it a promising approach for rapid and culture-free detection of Campylobacter in chicken products. 

   more » « less
3. Bacterial Wastewater-Based Epidemiology Using Surface-Enhanced Raman Spectroscopy and Machine Learning

   https://doi.org/10.1101/2024.07.22.604506  

   Herndon, Liam K; Zhang, Yirui; Safir, Fareeha; Ogunlade, Babatunde; Balch, Halleh B; Boehm, Alexandria B; Dionne, Jennifer A (July 2024, bioRxiv)

   Abstract Wastewater-based epidemiology (WBE) is a powerful tool for monitoring community disease occurrence, but current methods for bacterial detection suffer from limited scalability, the need fora prioriknowledge of the target organism, and the high degree of genetic similarity between different strains of the same species. Here, we show that surface-enhanced Raman spectroscopy (SERS) can be a scalable, label-free method for detection of bacteria in wastewater. We preferentially enhance Raman signal from bacteria in wastewater using positively-charged plasmonic gold nanorods (AuNRs) that electrostatically bind to the bacterial surface. Transmission cryoelectron microscopy (cryoEM) confirms that AuNRs bind selectively to bacteria in this wastewater matrix. We spike the bacterial speciesStaphylococcus epidermidis, Staphylococcus aureus, Serratia marcescens, andEscerichia coliand AuNRs into filter-sterilized wastewater, varying the AuNR concentration to achieve maximum signal across all pathogens. We then collect 540 spectra from each species, and train a machine learning (ML) model to identify bacterial species in wastewater. For bacterial concentrations of 10⁹cells/mL, we achieve an accuracy exceeding 85%. We also demonstrate that this system is effective at environmentally-realistic bacterial concentrations, with a limit of bacterial detection of 10⁴cells/mL. These results are a key first step toward a label-free, high-throughput platform for bacterial WBE. 

   more » « less
4. An Expanded Repertoire of tRNA Sources for Cell-Free Protein Synthesis

   https://doi.org/10.1101/2025.08.20.671396  

   Kalb, Evan M; Vincent, Russel M; Engelhart, Aaron E; Church, George M; Adamala, Katarzyna P (August 2025, bioRxiv)

   Abstract Cell-free expression systems (CFE) are flexible protein translation platforms that simplify the central dogma into an accessible reaction space. Within these systems, bulk transfer RNAs (tRNAs) are critical substrates which deliver amino acids to the elongating ribosome. For years, CFE systems were completed with commercially available tRNA isolated fromE. coliMRE600. All commercial sources of tRNA have since been discontinued, jeopardizing future work in all applications of cell-free translation. Here, we address this need by repurposing previously described tRNA isolation methods to produce tRNAs suitable for CFE applications. We isolated the tRNA pools ofE. colistrains A19, BL21(DE3), and Rosetta2 BL21(DE3), finding A19 tRNAs but not BL21(DE3) or Rosetta2 BL21(DE3) capable of robustin vitrotranslation. We determined the abundances of individual tRNAs using tRNA-seq, finding BL21(DE3) and Rosetta2 BL21(DE3) contained outsized abundances of several tRNAs, compromising translation activity. Using codon optimization strategies which align codon usage to tRNA abundance, we were able to mitigate the impact of misaligned tRNA abundances. We extended these studies toV. natriegens, a promising platform for synthetic biology and CFE. We find that neither exogenousV. natriegenstRNAs nor codon optimization are viable options to improve translation yields. Our work here highlights the importance of tRNA abundance within the context of CFE, and simultaneously addresses a critical challenge within cell-free translation. 

   more » « less
5. Deciphering transcript architectural complexity in bacteria and archaea

   https://doi.org/10.1128/mbio.02359-24  

   Mattick, John_S A; Bromley, Robin E; Watson, Kaylee J; Adkins, Ricky S; Holt, Christopher I; Lebov, Jarrett F; Sparklin, Benjamin C; Tyson, Tyonna S; Rasko, David A; Dunning_Hotopp, Julie C (October 2024, mBio)

   Parkhill, Julian (Ed.)

   ABSTRACT RNA transcripts are potential therapeutic targets, yet bacterial transcripts have uncharacterized biodiversity. We developed an algorithm for transcript prediction called tp.py using it to predict transcripts (mRNA and other RNAs) inEscherichia coliK12 and E2348/69 strains (Bacteria:gamma-Proteobacteria),Listeria monocytogenesstrains Scott A and RO15 (Bacteria:Firmicute),Pseudomonas aeruginosastrains SG17M and NN2 strains (Bacteria:gamma-Proteobacteria), andHaloferax volcanii(Archaea:Halobacteria). From >5 millionE. coliK12 and >3 millionE. coliE2348/69 newly generated Oxford Nanopore Technologies direct RNA sequencing reads, 2,487 K12 mRNAs and 1,844 E2348/69 mRNAs were predicted, with the K12 mRNAs containing more than half of the predictedE. coliK12 proteins. While the number of predicted transcripts varied by strain based on the amount of sequence data used, across all strains examined, the predicted average size of the mRNAs was 1.6–1.7 kbp, while the median size of the 5′- and 3′-untranslated regions (UTRs) were 30–90 bp. Given the lack of bacterial and archaeal transcript annotation, most predictions were of novel transcripts, but we also predicted many previously characterized mRNAs and ncRNAs, including post-transcriptionally generated transcripts and small RNAs associated with pathogenesis in theE. coliE2348/69LEEpathogenicity islands. We predicted small transcripts in the 100–200 bp range as well as >10 kbp transcripts for all strains, with the longest transcript for two of the seven strains being thenuooperon transcript, and for another two strains it was a phage/prophage transcript. This quick, easy, and reproducible method will facilitate the presentation of transcripts, and UTR predictions alongside coding sequences and protein predictions in bacterial genome annotation as important resources for the research community.IMPORTANCEOur understanding of bacterial and archaeal genes and genomes is largely focused on proteins since there have only been limited efforts to describe bacterial/archaeal RNA diversity. This contrasts with studies on the human genome, where transcripts were sequenced prior to the release of the human genome over two decades ago. We developed software for the quick, easy, and reproducible prediction of bacterial and archaeal transcripts from Oxford Nanopore Technologies direct RNA sequencing data. These predictions are urgently needed for more accurate studies examining bacterial/archaeal gene regulation, including regulation of virulence factors, and for the development of novel RNA-based therapeutics and diagnostics to combat bacterial pathogens, like those with extreme antimicrobial resistance. 

   more » « less